The long-term objective of this project is to obtain detailed biochemical information and understanding of the metabolism of acetate in the strictly anaerobic microorganism Methanosarcina barkeri. Specific goals are concerned with determination of structural and catalytic properties of a high molecular mass multienzyme complex that, as previous results of this project have demonstrated, catalyzes a critical, early event in acetate decomposition--namely, cleavage of the carbon-carbon bond of acetate (as acetyl-CoA). This project has shown that tetrahydrosarcinapterin (H4SPT) is required as a specific acceptor of the methyl group of acetyl-CoA, and that the carbonyl group is oxidized to carbon dioxide. Previous results have shown also that the reaction catalyzed by the enzyme complex is as follows: Acetyl-CoA + H4SPT + H2O -> CoA-SH + CH3-H4SPT + CO2 + 2[H] Currently, insight on the mechanism of this reaction has been gained by UV-visible and EPR spectroscopic analyses of reactions of the enzyme complex with CO and CO plus CH3-H4SPT. Catalysis of a related reaction in the direction of acetyl-CoA synthesis was shown to proceed as follows: CO + CH3-H4SPT + CoA-SH -> H4SPT + acetyl-CoA Partial reactions with CO alone caused stepwise reduction of enzyme Fe/S and corrinoid centers. Addition of CH3-H4SPT caused rapid methylation of the reduced corrinoid component. After previous reduction of the enzyme complex, further interaction with CO generated an EPR detectable adduct. Based upon observed quenching effects of CH3-H4SPT on the EPR radical signal, evidence was obtained supporting the hypothesis that the EPR signal may emanate from a one-carbon enzyme-bound precursor of the carbonyl group of acetyl-CoA. In the absence of CoASH, it is postulated that formation of an acetyl-enzyme intermediate is responsible for quenching of the EPR signal by CH3-H4SPT.